Can reduced consumption of gonadotrophins account for ovarian compensation in unilaterally ovariectomized, immature mice injected with gonadotrophins?

The effect of unilateral ovariectomy on ovulation rates in immature mice was studied. Ovulations were induced by injecting PMSG and hCG and their number was determined by counting tubal oocytes. A 2--3-fold increase in number of ovulations per ovary was observed after unilateral ovariectomy, and daily injections of progesterone abolished this ovulatory compensation. No significant increase in serum concentrations of immunoreactive FSH and LH was observed at 4, 8, 32 and 51 h after unilateral ovariectomy. Progesterone treatment lowered FSH levels at all times, while LH was unaffected. In intact mice, ovarian sensitivity to PMSG and hCG was not substantially affected by progesterone. Ovulatory compensation in immature gonadotrophin-injected mice appears to arise through a negative feedback mechanism and transiently increased secretion of pituitary gonadotrophin rather than through a greater utilization of a fixed amount of gonadotrophin.     

Different effects of amygdaloid lesions on compensatory ovarian hypertrophy in cyclic and prepubertal female rats.

Cyclic and 28-day-old immature female rats were hemiovariectomized and partly bilaterally lesioned in the cortical amygdaloid nucleus (CAN). The compensatory ovarian hypertrophy (COH) recorded in the adult females on the 8th day after hemicastration was completely prevented by the amygdaloid lesions. In contrast, damage to the CAN did not alter the ovarian weight in prepubertal females, although COH was also induced in these animals by unilateral ovariectomy.     

Uterine bioassay of tamoxifen,trioxifene and a new estrogen antagonist (LY117018) in rats and mice

Dose response uterotrophic and antiuterotrophic activity of antiestrogens was examined in immature rats, immature mice and adult ovariectomized mice. LY117018 was the most active antagonist and the least estrogenic, while tamoxifen induced the greatest uterine growth and the weakest antagonism. The reported estrogenic activity of tamoxifen in mice (1) was found to be related to maturity. All compounds caused uterotrophic changes in immature mice similar to those observed in immature rats. However, in adult mice tamoxifen was devoid of antagonism, and trioxifene was active only at a very high dose as both were extremely estrogenic in this model. LY117018 activity in adult mice was comparable to that observed in immature rats and mice. Results depict significant agonist and antagonist advantages of LY117018 over tamoxifen and trioxifene.     

Estrogen regulates pulmonary alveolar formation, loss, and regeneration in mice

Lung tissue elastic recoil and the dimension and number of pulmonary gas-exchange units (alveoli) are major determinants of gas-exchange function. Loss of gas-exchange function accelerates after menopause in the healthy aged and is progressively lost in individuals with chronic obstructive pulmonary disease (COPD). The latter, a disease of midlife and later, though more common in men than in women, is a disease to which women smokers and never smokers may be more susceptible than men; it is characterized by diminished lung tissue elastic recoil and presently irremediable alveolar loss. Ovariectomy in sexually immature rats diminishes the formation of alveoli, and estrogen prevents the diminution. In the present work, we found that estrogen receptor-alpha and estrogen receptor-beta, the only recognized mammalian estrogen receptors, are required for the formation of a full complement of alveoli in female mice. However, only the absence of estrogen receptor-beta diminishes lung elastic tissue recoil. Furthermore, ovariectomy in adult mice results, within 3 wk, in loss of alveoli and of alveolar surface area without a change of lung volume. Estrogen replacement, after alveolar loss, induces alveolar regeneration, reversing the architectural effects of ovariectomy. These studies 1) reveal estrogen receptors regulate alveolar size and number in a nonredundant manner, 2) show estrogen is required for maintenance of already formed alveoli and induces alveolar regeneration after their loss in adult ovariectomized mice, and 3) offer the possibility estrogen can slow alveolar loss and induce alveolar regeneration in women with COPD.     

Estrogen receptor-alpha regulates pulmonary alveolar loss and regeneration in female mice: morphometric and gene expression studies

Pulmonary alveoli, especially in females, are estrogen-responsive structures: ovariectomy in wild-type (WT) adult mice results in alveolar loss, and estradiol replacement induces alveolar regeneration. Furthermore, estrogen receptor (ER)-alpha and ER-beta are required for the developmental formation of a full complement of alveoli in female mice. We now show ovariectomy resulted in alveolar loss in adult ER-beta(-/-) mice but not in adult ER-alpha(-/-) mice. Estradiol treatment of ovariectomized ER-beta(-/-) mice induced alveolar regeneration. In ovariectomized WT mice, estradiol treatment resulted, within 1 h, in RNA-level gene expression supportive of processes needed to form an alveolar septum, e.g., cell replication, angiogenesis, extracellular matrix remodeling, and guided cell motion. Among these processes, protein expression supporting angiogenesis and cell replication was elevated 1 and 3 h, respectively, after estradiol treatment; similar findings were not present in either mutant. We conclude: 1) loss of signaling via ER-beta is not required for postovariectomy-induced alveolar loss or estradiol-induced regeneration; this indicates ER-alpha is key for estrogen-related alveolar loss and regeneration in adult female mice; 2) taken together with prior work showing that developmental formation of a full complement of alveoli requires ER-alpha and ER-beta, the present findings indicate the developmental and regenerative formation of alveoli are regulated differently, i.e., signaling for alveolar regeneration is not merely a recapitulation of signaling for developmental alveologenesis; and 3) the timing of estradiol-induced gene expression in lung supportive of processes required to form a septum differs between ovariectomized WT and ER-beta(-/-) mice.     

Long-term effects of ovariectomy on osteoporosis and obesity in estrogen-receptor-β-deleted mice

Untreated BERKO mice demonstrate few abnormalities in bone phenotype and recent ovariectomy has few effects on various bone characteristics in these mice. Long-term studies on the bone phenotype of intact and ovariectomized mice are unavailable. Using quantitative computed tomography (qCT), we determined various parameters of the metaphysis of the tibia in sham-ovariectomized (intact) and ovariectomized BERKO and wildtype mice. Body weight and estrogen-regulated fat were also measured. Mice underwent surgery (ovariectomy or sham) at 3 mo of age, and qCT analysis was performed every 2 to 4 mo until mice were 12 mo old. Ovariectomized wildtype mice gained body weight and their fat depot increased in size within 2 mo after ovariectomy. Obesity developed later in ovariectomized BERKO mice, which became significantly heavier than their wildtype counterparts. Ovariectomized wildtype mice lost trabecular density more rapidly than did ovariectomized BERKO mice, which did not show similar loss in trabecular density until at least 7 mo after ovariectomy. At the latest studied time point (9 mo after surgery), cortical area was significantly larger in ovariectomized BERKO mice than ovariectomized wildtype mice. The absence of ERβ in ovariectomized BERKO mice during the first 3 to 5 mo after ovariectomy had protective effects against obesity and trabecular rarification; this protective effect disappeared at later time points.     

Ovarian cyclicity and follicular recruitment in unilaterally ovariectomized mice

Halving the numbers of follicles in young adult mice by unilateral ovariectomy resulted in compensatory Graafian follicle growth with a reduction by about 25% of the expected number of oestrous cycles. The impact of the operation on the numbers and dynamics of preantral follicles during the first 2 months after ovariectomy was studied using a compartmental mathematical model to analyse differential follicle counts. There were changes in growth and/or death rates at all stages of follicle development, and the patterns emerging were time-dependent. The rate of follicle survival from the pool of unilaminar stages was paradoxically reduced, but those forming two granulosa cell layers continued to develop towards Graafian size. As the frequency of follicle death declined, the numbers of healthy large preantral and antral stages in unpaired ovaries rose to approach those in pairs of age-matched control ovaries, suggesting that follicles otherwise undergoing atresia were being rescued. In the long-term, follicle dynamics after unilateral ovariectomy at young ages did not appear to compromise fecundity seriously.     

Glycoconjugates on corneal epithelial surface: effect of neuraminidase treatment

The purpose of this study was to develop a procedure to quantitatively examine corneal epithelial apical cell membrane-associated glycoconjugates. Saccharide moieties on young, mature, and aged corneal epithelial cells were detected and localized in corneas of immature and adult mice by using colloidal gold-labeled lectins and transmission electron microscopy (TEM). In general, dense binding to the corneal epithelial apical surface cell membranes with wheat germ agglutinin (WGA) was seen in the adult, whereas the immature cornea bound less WGA-gold. Neuraminidase digestion decreased binding of the conjugate on epithelial plasma membranes of young and mature cells in adult cornea. Lectin-gold binding was decreased in the immature cornea on mature and aged cells. WGA-gold binding after neuraminidase was elevated on young cells of immature and on aged cells of adult animals. No binding of peanut agglutinin (PNA) or horse gram agglutinin (DBA) to the corneal epithelial surface was seen in animals of either age. After neuraminidase digestion, PNA binding sites were exposed only on the adult corneal surface. These data suggest that a terminal trisaccharide sequence, sialic acid-galactose beta(1----3)-N-acetylgalactosamine, is present at the adult corneal surface but is absent or at undetectable levels at the corneal surface of the immature animal. These data may be of significance in light of the dissimilar pattern of P. aeruginosa recognition and binding to the immature vs adult corneal epithelium.     

Inhibitory action of leptin on early follicular growth differs in immature and adult female mice.

In order to investigate the action of leptin on early follicular growth, preantral follicles, 95-115 microm in diameter were mechanically isolated from the ovaries of BDF1 hybrid immature (11-day-old) and adult (8-wk-old) mice, and cultured for 4 days in vitro. Follicular growth was assessed by daily changes in follicular diameter and by the amount of estradiol and immunoreactive (IR)-inhibin released into the culture medium at Day 4. Preantral follicles from immature mice showed a significant development in follicular growth as a result of stimulation by GH (1 mIU/ml), insulin-like growth factor (IGF)-I (100 ng/ml) + FSH (100 mIU/ml), and GH (1 mIU/ml) + FSH (100 mIU/ml). Although leptin at concentrations of 1-1000 ng/ml did not have any significant effect on follicular growth stimulated by IGF-I or GH, it significantly inhibited follicular growth in a dose-related manner when follicles were stimulated by IGF-I + FSH and GH + FSH, respectively, suggesting that leptin attenuated the additive effect of FSH. On the other hand, preantral follicles from adult mice were cultured in the presence of FSH, and FSH-dependent follicular growth was inhibited by leptin in a dose-related manner. Because FSH stimulates cAMP production, we investigated the involvement of cAMP in the inhibitory mechanisms of leptin. Preantral follicles from immature and adult mice were cultured in the presence of either 8-Br-cAMP or forskolin. Both 8-Br-cAMP and forskolin significantly increased follicular diameter and hormone secretion in both immature and adult mice. However, 8-Br-cAMP and forskolin-stimulated follicle growth and hormone secretion were significantly inhibited in immature mice by coadministration of leptin, whereas growth of preantral follicles from adult mice was not inhibited by addition of leptin to cultures. These results indicate that leptin causes an inhibitory effect on the early follicular development of both immature and adult mice, but the inhibitory mechanisms of leptin are different.     

Changes in the concentration of high-affinity oestradiol receptors in rat uterine supernatant preparations during the oestrous cycle, pseudopregnancy, pregnancy, maturation and after ovariectomy

A quantitative method was used to determine the concentration of high-affinity oestradiol-receptor sites in rat uterine supernatant preparations under various physiological conditions. Cyclic changes in concentration were observed during the oestrous cycle, with a maximum occurring in late dioestrus. The changes followed a similar pattern in endometrium and myometrium, although concentrations were higher in the former. In pseudopregnancy the concentration was initially low, rising to a maximum on the tenth day. In early pregnancy a high concentration of receptor was found to be associated with the developing placenta, but this declined in later stages of pregnancy. After ovariectomy or combined ovariectomy and adrenalectomy the receptor concentration remained at a constant low value that could be increased by treatment with oestradiol. The receptor concentration was considerably higher in immature than in adult uteri.     

Effect of unilateral ovariectomy and subsequent ovum implantation in mice.

Unilateral ovariectomy (ULO) was done on any stage of the cycle and the animals were mated within day 1 to day 21 to observe the acute and long term effect of ULO on ovum implantation. Implantation reduced in proportion to single ovary if the animals were mated within 24 hr of ULO. Increase in ovarian weight along with an increase in implantation number continued in mated mice and reached at peak on day 19-21 of ULO (sacrificed after 6 days i.e., 25-27 days of ULO). After ULO the remaining ovary compensated within day 5-6 of ULO even during pregnancy. Ovarian histology showed stimulation of small antral follicles in mice mated on day 3 of ULO (sacrificed after 6 days i.e., day 9 of ULO) along with a decrease of large antral follicles and pre-antral follicles. Preantral follicles were at peak on day 12-14. Large antral follicles attained a peak on day 4 which slowly decreased. The occurrence of implantation in such ULO conditions are discussed.     

Immature hematopoietic stem cells undergo maturation in the fetal liver

Hematopoietic stem cells (HSCs), which are defined by their capacity to reconstitute adult conventional mice, are first found in the dorsal aorta after 10.5 days post coitus (dpc) and in the fetal liver at 11 dpc. However, lympho-myeloid hematopoietic progenitors are detected in the dorsal aorta from 9 dpc, raising the issue of their role in establishing adult hematopoiesis. Here, we show that these progenitors are endowed with long-term reconstitution capacity, but only engraft natural killer (NK)-deficient Rag2γc(-/-) mice. This novel population, called here immature HSCs, evolves in culture with thrombopoietin and stromal cells, into HSCs, defined by acquisition of CD45 and MHC-1 expression and by the capacity to reconstitute NK-competent mice. This evolution occurs during ontogeny, as early colonization of fetal liver by immature HSCs precedes that of HSCs. Moreover, organ culture experiments show that immature HSCs acquire, in this environment, the features of HSCs.     

Subventricular zone astrocytes are neural stem cells in the adult mammalian brain.

Neural stem cells reside in the subventricular zone (SVZ) of the adult mammalian brain. This germinal region, which continually generates new neurons destined for the olfactory bulb, is composed of four cell types: migrating neuroblasts, immature precursors, astrocytes, and ependymal cells. Here we show that SVZ astrocytes, and not ependymal cells, remain labeled with proliferation markers after long survivals in adult mice. After elimination of immature precursors and neuroblasts by an antimitotic treatment, SVZ astrocytes divide to generate immature precursors and neuroblasts. Furthermore, in untreated mice, SVZ astrocytes specifically infected with a retrovirus give rise to new neurons in the olfactory bulb. Finally, we show that SVZ astrocytes give rise to cells that grow into multipotent neurospheres in vitro. We conclude that SVZ astrocytes act as neural stem cells in both the normal and regenerating brain.     

Interleukin-6 expression during normal maturation of the mouse testis

In this study, we examined the cellular origin and the expression levels of interleukin-6 (IL-6) during normal maturation of mouse testis. The levels of IL-6 (protein and mRNA) were higher in testicular homogenates of sexually immature than mature mice. Immunohistochemical staining of testicular tissues of sexually immature and adult mice show that testicular germ cells, at different stages of differentiation, Leydig cells/interstitial cells and peritubular cells express IL-6. Our results demonstrate, for the first time, overexpression of IL-6 in testicular tissues of immature mice, as compared to mature mice, as well as the expression of IL-6 in germ cells of testicular tissues of adult and sexually immature mice. Thus, our results may indicate the involvement of the endocrine system (gonadotropins and testosterone) in the regulation of IL-6, which is involved in the regulation of testicular development, functions and spermatogenesis.     

Over expression of interleukin-1alpha,interleukin-1beta and interleukin-1 receptor antagonist in testicular tissues from sexually immature mice as compared to adult mice

The levels of IL-1alpha, IL-1beta and IL-1Ra were higher in homogenates of testicular tissue from sexually immature than those from mature mice. Immunohistochemical staining of testicular tissues from sexually immature and adult mice show that differentiated germ cells express higher levels of IL-1alpha compared to Sertoli cells and Leydig cells/interstitial cells. Peritubular cells of sexually immature and adult mice did not express IL-1alpha. Testicular tissue cells of adult mice showed high levels of expression of IL-1beta, mainly in the cytoplasm and nucleus of the spermatogonia and in spermatocytes. Sertoli cells and Leydig/interstitial cells were also highly stained for IL-1beta. However, peritubular cells did not express IL-1beta. On the other hand, testicular tissue cells from sexually immature mice, showed high levels of IL-1beta, mainly in spermatocytes. Spermatogonia showed low levels of IL-1beta expression. Also, high levels of IL-1beta expression were detected in Leydig/interstitial cells. Peritubular cells clearly showed IL-1beta expression. Testicular tissue cells from adult mice, showed IL-1Ra expression in spermatogonia, Sertoli and Leydig/interstitial cells. IL-1Ra expression was clearly present in the Golgi apparatus of spermatogonia and Sertoli cells. However, peritubular cells did not show IL-1Ra expression. Testicular tissue cells from sexually immature mice, also showed high levels of IL-1Ra expression mainly in the cytoplasm and nucleus of the spermatogonia and Sertoli cells. In addition, Leydig/interstitial cells and peritubular cells also expressed IL-1Ra. Our results demonstrate, for the first time, the expression of IL-1beta in germ and Sertoli cells, and IL-1Ra in Leydig/interstitial cells of testicular tissues from adult and sexually immature mice, under in vivo conditions. In addition, the relative elevated levels of the IL-1 system in the testis of immature mice compared to mature mice may indicate its involvement in the spermatogenesis.     

Alpha-fetoprotein can regulate growth in the uterus of the immature and adult ovariectomized mouse

This is a report of development of (1) a 3-day immature mouse bioassay for alpha-fetoprotein (AFP) to increase the working range in uterine wet weights over-coming seasonality, and (2) a bioassay for AFP performed with ovariectomized adult mice to increase sensitivity. Mouse AFP was isolated from amniotic fluid and purified by polyacrylamide gel electrophoresis followed by Blue Sepharose chromatography. The uterine growth evoked by the injection of 1.0 microgram AFP together with excess molar oestradiol (0.5 microgram) over a 23-h period was compared in immature ovariectomized adult Nylar mice. The 3-day assay with immature mice was rendered usable in any season, with sensitivity comparable to the 1-day assay. Increased sensitivity, however, was demonstrated by utilization of AFP in a 1-day assay with the adult ovariectomized mouse.     

Sexual immaturity and maternal age: incidence of aneuploidy and polyploidy in first-cleavage mouse embryos

The influence of maternal age on the incidence of aneuploidy and polyploidy was studied, using C57Bl/6J X CBA/Ca hybrid mice, including immature females, as gamete donors. The age of the females ranged from 3.5-4 wk (immature or prepubertal), to 10-12 wk (young adults), to 24-28 wk (aged females). Ovulation was induced with gonadotrophins, and the differential condensation of paternal and maternal chromosomes was used to elucidate the origin of chromosome abnormalities in first-division metaphase plates. The results indicated a high incidence of aneuploid oocytes in immature and older female mice, as compared to young adult females. Eggs of immature female mice underwent polyspermic fertilization more often than those of young adults and older females, and the production of diploid oocytes was more frequent in immature females than in the other age groups.     

Morphological changes in the submandibular glands and in the X zone of the adrenal gland following ovariectomy in mice

Summary Morphological changes in submandibular glands of female mice following ovariectomy were studied morphometrically by light microscopy and ultrastructurally by electron microscopy. The X zone of the adrenal gland was examined in order to assess possible changes that might be expected to occur after ovariectomy.In submandibular glands, 1 to 4 weeks after ovariectomy, no changes were observed in percentages of the acinar, intercalated duct, and granular convoluted tubular areas occupying photomicrographs. However, an increase in the granular content of both intercalated duct and granular convoluted tubular cells was recognized. By contrast, the glandular picture 4 months after ovariectomy changed remarkably, resembling that of the male mouse both morphometrically and in terms of fine structure. In the adrenal cortex of control female mice, the X zone became thinner with aging. As compared with this, the X zone of ovariectomized mice at any time after the operation was thicker than that of controls.These observations suggest that the absence of ovarian hormones in the ovariectomized mouse may lead to prolonged functioning of X zone cells, which in turn may cause masculinization of the submandibular gland.     

The effect of donor age on progression of spermatogenesis in canine testicular tissue after xenografting into immunodeficient mice

The objective of this study was to examine the effect of donor age on progression of spermatogenesis in dog (Canis lupus familiaris) testis tissue after xenografting. In Experiment 1, canine testes were obtained by surgical castration. Based on developmental pattern of spermatogenesis at the time of grafting, donors were categorized as immature, young, and adult (<4, 4 to 6, and >6 mo old, respectively). Fragments of testis tissue were implanted subcutaneously on the back of immunodeficient mice; xenografts were retrieved and analyzed 4, 6, or 8 mo later. At 4 mo postgrafting, immature and young groups had higher graft recovery rates, graft weights, vesicular gland indices, seminiferous tubule numbers, and larger seminiferous tubular diameters compared with those of adult donor xenografts. At 8 mo postgrafting, immature donor xenografts had maintained growth and development as exhibited by greater graft weights, vesicular gland indices, seminiferous tubule numbers, and tubular diameters compared with those of adult donor xenografts. At this time point, growth and development of xenografts did not differ between immature and young donors, whereas those from young donors had greater seminiferous tubule numbers and diameters compared with those of adult donor xenografts. Elongated spermatids were the most advanced germ cell type present at 4 and 8 mo postgrafting in xenografts of immature age groups. In Experiment 2, the longer-term efficiency of spermatogenesis and the potential sperm production in xenografts from immature donor dogs were determined. Testis tissue from 2-mo-old donor dogs were grafted into recipient mice, and xenografts were retrieved after 13 mo. Complete spermatogenesis was present in 5 of 29 recovered xenografts, with isolation of fully formed sperm (up to 36.3 × 10⁶ per gram tissue). In conclusion, immature and young donors (<6 mo of age) were the most promising donors for dog testis tissue xenografting. This strategy may offer an alternative for male germ-line preservation for canids that die prematurely or must be castrated before maturation.     

Re-examination on the sex-influenced esterase (ESSI) in rat serum

The sex-limitation of sex-influenced esterase (ESSI) in serum of rats carrying Es-Sia allele was re-examined. ESSI was detected in immature males and females, and orchiectomized rats as well as mature females whereas ESSI in normal males rapidly disappeared with puberty. The rats orchiectomized at weaning temporarily lost ESSI around the age of sexual maturation, thereafter, ESSI reappeared. When orchiectomized rats were administered testosterone, synthesis of ESSI was suppressed as in normal adult males. Effect of ovariectomy was not recognized. The exceptional strain named SI 3 of which normal mature males have a significant level of ESSI has been established, although the level in the males is lower than that in the females of the same strain.